# Immunomodulatory Effect of Artemisia annua L. Water Extract on Meat-Type Sheep via Activating TLR4/NF-κB Signaling Pathways

## TL;DR

This study shows that a water extract from Artemisia annua boosts sheep immunity by activating the TLR4/NF-κB pathway, improving health in intensive farming.

## Contribution

The study demonstrates the immunomodulatory effect of Artemisia annua water extract in sheep via the TLR4/NF-κB pathway, offering a novel functional feed supplement.

## Key findings

- WEAA dose-dependently increased immunoglobulins and cytokines in serum, liver, and spleen tissues.
- WEAA upregulated TLR4/NF-κB pathway genes and improved immune function in sheep.
- The optimal WEAA dose was determined to be 1000 mg/kg for feed supplementation.

## Abstract

Due to the rapid advancement of intensive meat-type sheep farming in China, the industry is confronting a series of stress challenges, including high-density rearing environments, environmental pollution, imbalanced feed nutrition, and frequent disease outbreaks. These stressors may lead to oxidative imbalance and immune dysfunction in sheep, thereby adversely affecting livestock health and production performance. Artemisia annua L. and its extracts contain various bioactive compounds and exhibit immunomodulatory effects, thereby potentially enhancing the health status of animal organisms. Based on the abovementioned potential, this study aimed to comprehensively evaluate the immunomodulatory function of water extract of Artemisia annua L. (WEAA) in the serum, liver, and spleen of sheep and explore the underlying mechanistic pathways via in vivo and in vitro experiments. The results showed that dietary supplementation of WEAA enhanced sheep’s immune indicators by upregulating TLR4/NF-κB pathway genes, thereby coordinately regulating humoral and innate immunity, thereby improving the immune indices of sheep.

This experiment was designed to systematically evaluate the immunomodulatory effect of water extract of Artemisia annua L. (WEAA) on sheep, both in vivo and in vitro, and to determine the involvement of the TLR4/NF-κB signaling pathway in mediating these effects. In experiment 1, 32 female sheep (Dorper × Han, 3 months old, 24 ± 0.09 kg each) were designated to 4 groups, with each group receiving a basal diet supplemented with, respectively, 0 (control group), 500, 1000, and 1500 mg/kg WEAA. The serum, liver, and spleen immune indicators and related gene expressions were measured. In experiment 2, the peripheral blood lymphocytes (PBLs) were processed with WEAA (0, 25, 50, 100, 200, and 400 μg/mL), with six replicates assigned to each concentration group, then the cell viability, immune function, and related gene expressions were measured, and the optimal concentration of WEAA was determined. In experiment 3, the experimental groups consisted of PBLs subjected to treatments with or without PDTC (NF-κB inhibitor) and with or without WEAA, forming four distinct treatment groups (six replicates/group): PDTC(−)/WEAA(−) group, PDTC(−)/WEAA(+) group, PDTC(+)/WEAA(−) group and PDTC(+)/WEAA(+) group. The immune indexes and TLR4/NFκB pathway related indexes were determined. The results were as follows: WEAA dose-dependently enhanced the content of immunoglobulins (IgA, IgG, IgM) and cytokines (IL-1β, IL-2, IL-4) in the serum, liver, and spleen tissues, among which IgA, IgG, and IL-4 were the most significantly affected core indicators (p < 0.05). Meanwhile, WEAA dose-dependently upregulated the expression of TLR4/NF-κB pathway-related genes (TLR4, IKKβ, IκBα, NF-κBp65) and their downstream cytokine-related genes (IL-1β, IL-4) in liver and spleen tissues (p < 0.05). Of these genes, liver IL-4, IκBα, and spleen IL-4 were the most prominently regulated core genes (p < 0.05), The optimal supplementary dose of WEAA was determined to be 1000 mg/kg. In addition, adding 100 μg/mL WEAA to the culture medium of PBLs significantly enhanced immune function and cell viability. The underlying mechanism involved the TLR4/NF-κB pathway; that is to say, WEAA enhanced sheep’s immune indicators by upregulating TLR4/NF-κB pathway genes, thereby coordinately regulating humoral and innate immunity, thereby improving the immune indices of sheep. This study provided compelling experimental support for the prospective utilization of WEAA as a functional feed supplement in intensive meat-type sheep production systems.